History
A 12-year-old spayed female Labrador Retriever mix weighing 38.3 kg (84.3 lb) was referred to the University of Georgia College of Veterinary Medicine because of multifocal nonpruritic raised cutaneous lesions, lethargy, and anorexia of approximately 1 week's duration. The dog was treated initially by the referring veterinarian with an injection (route unknown) of diphenhydramine and a 5-day course of orally administered corticosteroids. At a recheck examination, the dog had had no response to treatment.
Clinical and Clinicopathologic Findings
A CBC and serum biochemical panel performed by the referring veterinarian revealed thrombocytopenia (38 × 109 platelets/L; reference interval, 200 × 109 platelets/L to 500 × 109 platelets/L) and lymphopenia (0.81 × 109 lymphocytes/L; reference interval, 1 × 109 lymphocytes/L to 4.8 × 109 lymphocytes/L). Because of ongoing anorexia, abdominal radiography was performed by the referring veterinarian and revealed possible splenomegaly. At the referral examination, the dog was lethargic but alert and responsive. Mild hyperthermia (39.28°C [102.7°F]), moderate tachycardia (160 beats/min), and mild tachypnea with increased abdominal effort were noted on physical examination. Examination of the oral cavity revealed moderate dental calculus accumulation and multiple raised, erythematous lesions on the mucous membranes and mucocutaneous junctions. On thoracic auscultation, wheezing was evident. Palpation revealed left popliteal and bilateral mandibular lymphadenopathy. A detailed dermatologic examination revealed multiple, nonpainful, nonpruritic, erythematous, raised nodules on mucous membranes, at mucocutaneous junctions, on distal portions of the limbs, on the ventral aspect of the abdomen, and along the dorsum; some nodules were associated with focal ulceration (Figure 1). Thoracic radiography revealed a moderately enlarged sternal lymph node and a mild, diffuse bronchial pattern. Abdominal ultrasonography revealed a diffusely, mildly hypoechoic liver with rounded margins, multifocal irregularly shaped hypoechoic splenic nodules, a left adrenal gland nodule, left medial iliac and gastric lymphadenopathy, hyperechoic mesentery in the cranial portion of the abdomen, and a heterogeneously hyperechoic right limb of the pancreas. The splenomegaly suspected during the first examination was not identified. In addition, multiple small ovoid nodules were detected in the ventral portion of the body wall.
Photographs of the mouth (A), ventral aspect of the abdomen (B), and left antebrachium (C) of a dog that was referred because of multifocal nonpruritic raised cutaneous lesions, lethargy, and anorexia of approximately 1 week's duration. Multifocal raised lesions were present on the mucous membranes, mucocutaneous junctions, and skin.
Citation: Journal of the American Veterinary Medical Association 249, 12; 10.2460/javma.249.12.1375
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Histopathologic and Cytologic Findings
Fine-needle aspirate specimens of the cutaneous masses and the mandibular lymph nodes were obtained. Microscopic examination of the fine-needle aspirate specimens of the cutaneous masses revealed high numbers of individualized round cells that were 15 to 30 μm in diameter with a high nuclear-to-cytoplasmic ratio (Figure 2). The cytoplasm of the cells was scant to moderate and deeply basophilic with several small, round, clear vacuoles and occasionally an eosinophilic dusting. The nucleus was round to oval to irregular, was often eccentrically located, and had finely stippled chromatin with variable numbers of prominent nucleoli. Scattered, atypical mitotic figures were also observed. Anisocytosis and anisokaryosis were pronounced, and micronuclei were occasionally present. Low numbers of well-differentiated mast cells (comprising < 1% of the nucleated cells) and rare cytoplasmic fragments (resembling lymphoglandular bodies) were present in the background. Microscopic examination of the fine-needle aspirate specimens of the mandibular lymph nodes revealed the resident lymphoid population had been almost completely replaced by cells with the same morphologic characteristics as those found in the skin masses. These cells had occasionally undergone leukophagocytosis. A diagnosis of round cell neoplasia was made, and given the high nuclear-to-cytoplasmic ratio and the rare lymphoglandular bodies seen in the skin masses, lymphoid neoplasia was considered the primary differential diagnosis. However, the cytoplasmic vacuolation and the prominent nuclear variability were atypical for lymphoma. Undifferentiated mast cell tumor, histiocytic neoplasia, and amelanotic melanoma were also considered as differential diagnoses but were deemed less likely.
Photomicrograph of a fine-needle aspirate specimen obtained from a representative skin mass on the dog in Figure 1. There are several individualized round cells with a high nuclear-to-cytoplasmic ratio and pronounced anisocytosis and anisokaryosis. Atypical mitotic figures can be observed (arrows). Romanowsky stain; bar = 20 μm.
Citation: Journal of the American Veterinary Medical Association 249, 12; 10.2460/javma.249.12.1375
On the basis of the cytologic findings and the presence of multiple masses detected via abdominal ultrasonography, metastatic neoplasia was suspected. Owing to the dog's poor prognosis, the owner elected euthanasia (by means of IV injection of pentobarbital solution). Although a full necropsy was declined, owner consent was given for multiple skin biopsies.
Histologic examination of the skin biopsy specimens revealed that proliferating neoplastic cells had effaced areas of the dermis and subcutis and extended to the epidermis (Figure 3). The neoplastic masses were poorly demarcated, infiltrative, and composed of sheets and cords of neoplastic cells sustained by a fine fibrovascular stroma. Cells were round to polygonal and approximately 10 to 20 μm in diameter and had a high nuclear-to-cytoplasmic ratio. The nucleus was round to oval to irregular and had an open, vesicular chromatin pattern with 1 to multiple nucleoli. Anisocytosis and anisokaryosis were considered moderate to marked. There were 75 mitotic figures/10 hpf (40×), and numerous neoplastic cells were seen in vessels in proximity of the masses. Low numbers of neutrophils were admixed with the neoplastic cells and, occasionally, the epidermis overlying the masses was ulcerated. The histologic appearance of the neoplastic masses was most consistent with lymphoma.
Photomicrographs of a section of a representative haired skin mass on the dog in Figure 1. Areas of subcutaneous adipose tissue have been replaced by sheets and cords of neoplastic cells that extend to the epidermis. H&E stain; bar = 200 μm. Inset—Vascular invasion by neoplastic cells is present. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 249, 12; 10.2460/javma.249.12.1375
Immunohistochemical and Molecular Findings
To further characterize the neoplastic cells, immunohistochemical stains for CD3, CD79a, CD18, CD20, CD21, granzyme B, and melan-A were performed on formalin-fixed, paraffin-embedded skin biopsy tissue samples. Neoplastic cells were immunoreactive for CD45 and granzyme B (Figure 4). Rare neoplastic cells—particularly the neoplastic cells within vessels adjacent to the neoplasm—were strongly immunoreactive for CD18. Approximately 30% of cells expressed only punctate reactivity for CD18; the remaining cells lacked immunoreactivity for CD18. Neoplastic cells lacked immunoreactivity for CD3, CD79a, CD20, CD21, and melan-A.
Photomicrograph of a section of a representative haired skin mass on the dog in Figure 1. The neoplastic cells infiltrating the skin have marked cytoplasmic reactivity with antibodies against granzyme B. Immunohistochemical stain for granzyme B; bar = 100 μm.
Citation: Journal of the American Veterinary Medical Association 249, 12; 10.2460/javma.249.12.1375
Deoxyribonucleic acid was extracted from formalin-fixed, paraffin-embedded material via standard methods.a A PCR assay for antigen receptor rearrangement was performed with T-cell receptor γ (TCRγ) primer sets, as previously described.1 The PCR products were visualized with capillary electrophoresis. A monoclonal TCRγ rearrangement was detected.
Morphologic Diagnosis and Case Summary
Morphologic diagnosis and case summary: disseminated nonepitheliotropic cutaneous T-cell lymphoma in a dog.
Comments
Nonepitheliotropic cutaneous T-cell lymphoma (NE-CTCL) is an uncommon neoplasm in dogs.2 Cutaneous nonepitheliotropic lymphoma may be primary or may develop in conjunction with disseminated lymphoma and is mainly of T-cell origin.3 When the neoplastic cells have definitive CD3 expression, the diagnosis of NE-CTCL is readily made. However, the diagnosis can be complicated by the absence of CD3 expression,2 as for the dog of the present report. Neoplasia of natural killer (NK)-cell origin was suspected after immunohistochemical analysis, given the lack of immunoreactivity for T- and B-lymphocyte markers. More specifically, neoplastic cells did not react with antibodies against CD3 (a T-lymphocyte marker), CD79a (a surface marker present on both immature and mature B lymphocytes, including plasma cells), and CD20 and CD21 (markers for mature B lymphocytes). Expression of CD18 in histiocytic, dendritic, and myeloid cells is consistent, whereas expression in lymphocytes and NK cells is variable and may be undetectable.4 CD18 is a nonspecific antigen involved in leukocyte adhesion, and strong CD18 immunoreactivity was occasionally seen in neoplastic cells inside vessels in the case described in the present report. The propensity of neoplastic cells in vessels to express CD18 may be explained by the need of these cells to extravasate into peripheral tissues and thus to express leukocyte adhesion integrins.5 The loss of CD18 expression in extravasated neoplastic cells may be explained by a lack of need of integrins. Melan-A is a cytoplasmic protein specific for cells with melanocytic differentiation that is often used for melanoma diagnosis.6 For the dog of the present report, melanoma was excluded as a differential diagnosis owing to the lack of immunoreactivity for this marker in skin biopsy specimens. CD45 is a common leukocyte antigen, whereas granzyme B is a protein present in the granules of cytotoxic T cells and NK cells.7
Loss of or aberrant antigen expression is a common feature of lymphoma in both humans and dogs, and although it can be a useful marker of malignancy, it may cause difficulties in formulating a definitive diagnosis.8,9 For the dog of this report, demonstration of a clonal TCRγ rearrangement was pivotal in confirming the T-cell origin of the neoplastic cells and, ultimately, in making a final diagnosis of NE-CTCL. Tumors of true NK-cell origin, as suggested by the staining pattern, would not have a TCRγ rearrangement.10
Single to multiple cutaneous masses in canine cases of NE-CTCL have been previously described.2,11 Nonepitheliotropic cutaneous T-cell lymphoma-related masses are typically nonpruritic but can be associated with alopecia and ulceration.2,11 The Labrador Retriever mix dog of this report had nonpruritic, diffuse cutaneous lesions as well as lesions at the mucous membranes and mucocutaneous junctions. Abdominal ultrasonography revealed multiple nodules in the spleen, left adrenal gland, and abdominal wall, suggestive of metastasis to the internal organs. However, it cannot be determined whether the dog had primary cutaneous neoplasia with widespread metastasis versus systemic neoplasia with secondary cutaneous involvement. This case illustrates the growing importance of molecular diagnostic testing in the diagnosis of lymphoid neoplasia in dogs.
Acknowledgments
The authors thank Drs. Elizabeth Howerth and Pauline Rakich for assistance in the diagnostic process, Mrs. Abbie Butler for assistance with immunohistochemical staining, and Mr. Kenny Williamson and Mrs. Dawn Dunbar for assistance with images.
Footnotes
QIAamp DNA FFPE Tissue kit, Qiagen Ltd, Crawley, West Sussex, England.
References
1. Chaubert P, Baur Chaubert AS, Sattler U, et al. Improved polymerase chain reaction-based method to detect early-stage epitheliotropic T-cell lymphoma (mycosis fungoides) in formalin-fixed, paraffin-embedded skin biopsy specimens of the dog. J Vet Diagn Invest 2010; 22: 20–29.
2. Moore PF, Affolter VK, Keller SM. Canine inflamed nonepitheliotropic cutaneous T-cell lymphoma: a diagnostic conundrum. Vet Dermatol 2013; 24: 204–211, e44–e45.
3. Gross TL, Ihrke PJ, Walder EJ, et al. Lymphocytic tumors. In: Skin diseases of the dog and cat: clinical and histopathologic diagnosis. 2nd ed. Oxford, England: Blackwell Science, 2010;882–888.
4. Fernandez NJ, Kidney BA, Jackson ML, et al. Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors. Vet Pathol 2005; 42: 437–445.
5. Grabbe S, Varga G, Beissert S, et al. Beta2 integrins are required for skin homing of primed T cells but not for priming naive T cells. J Clin Invest 2002; 109: 183–192.
6. Ramos-Vara JA, Miller MA. Immunohistochemical identification of canine melanocytic neoplasms with antibodies to melanocytic antigen PNL2 and thyrosinase: comparison with Melan A. Vet Pathol 2011; 48: 443–450.
7. Lane LV, Allison RW, Rizzi TR, et al. Canine intravascular lymphoma with overt leukemia. Vet Clin Pathol 2012; 41: 84–91.
8. Rodriguez-Abreu D, Filho VB, Zucca E. Peripheral T-cell lymphomas, unspecified (or not otherwise specified): a review. Hematol Oncol 2008; 26: 8–20.
9. Gelain ME, Mazzilli M, Riondato F, et al. Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry. Vet Immunol Immunopathol 2008; 121: 179–188.
10. van Dongen JJ, Langerak AW, Brüggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–2317.
11. Affolter VK, Gross TL, Moore PF. Indolent cutaneous T-cell lymphoma presenting as cutaneous lymphocytosis in dogs. Vet Dermatol 2009; 20: 577–585.
